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Crystal structures of Mmm1 and Mdm12–Mmm1 reveal PNAS PLUS mechanistic insight into trafficking at ER-mitochondria contact sites

Hanbin Jeonga,b, Jumi Parka,b, Youngsoo Junb,c, and Changwook Leea,b,d,1

aDepartment of Biological Sciences, School of Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea; bCell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea; cSchool of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea; and dCenter for Genome Integrity, Institute for Basic Science, Ulsan 44919, Republic of Korea

Edited by Peter J. Novick, University of California, San Diego, La Jolla, CA, and approved September 28, 2017 (received for review September 7, 2017) The (ER)-mitochondria encounter structure ERMES complex and regulates its size and number (14–16). (ERMES) comprises mitochondrial distribution and morphology 12 ERMES components are also regulated by Rsp5 E3 ubiquitin li- (Mdm12), maintenance of mitochondrial morphology 1 (Mmm1), gase, and ubiquitination is required for efficient mitophagy (17). Mdm34, and Mdm10 and mediates physical membrane contact sites Accumulated evidence suggests that Mdm12, Mmm1, and and nonvesicular trafficking between the ER and mitochon- Mdm34 share a -like mitochondrial lipid-binding dria in . Herein, we report two crystal structures of the (SMP) domain (7, 18–20), suggesting that the ERMES synaptotagmin-like mitochondrial lipid-binding protein (SMP) do- complex not only tethers two connecting membranes but also acts main of Mmm1 and the Mdm12–Mmm1 complex at 2.8 Å and 3.8 Å as a transfer vehicle to exchange between the ER resolution, respectively. Mmm1 adopts a dimeric SMP structure and mitochondria (21). Indeed, ERMES mutants have an altered augmented with two extra structural elements at the N and C (PS)-to- (PE) con- termini that are involved in tight self-association and phospholipid version rate (13, 22), suggesting that the ERMES complex might be coordination. Mmm1 binds two phospholipids inside the hydro- critically involved in phospholipid trafficking at ER-mitochondria phobic cavity, and the phosphate ion of the distal phospholipid contact sites, although its direct involvement in converting PS to is specifically recognized through extensive H-bonds. A positively PE still remains contentious (23). Recent studies have highlighted charged concave surface on the SMP domain not only mediates ER alternative lipid trafficking pathways involving vacuoles, which re- membrane docking but also results in preferential binding to glyc- ciprocally supply mitochondria with phospholipids (24–26). Fur- erophospholipids such as (PC), phosphatidic thermore, the ER complex (EMC) comprising (PA), (PG), and phosphatidylserine (PS), conserved Emc1–Emc6 performs a comparable role in some of which are substrates for lipid-modifying in mi- lipid transfer from the ER to mitochondria by mediating tethering – tochondria. The Mdm12 Mmm1 structure reveals two Mdm12s between these (26). In addition to lipid trafficking, other binding to the SMP domains of the Mmm1 dimer in a pairwise functions of the ERMES complex have been reported, including head-to-tail manner. Direct association of Mmm1 and Mdm12 gen- mitochondrial protein assembly (27) and import (28), maintenance erates a 210-Å-long continuous hydrophobic tunnel that facili- – of mitochondrial DNA (15, 29, 30), mitochondria inheritance (31), tates phospholipid transport. The Mdm12 Mmm1 complex binds and mitophagy (17, 32–34). all except for phosphatidylethanolamine (PE) in vitro. Significance Mmm1 | Mdm12–Mmm1 complex | ERMES | phospholipid trafficking | The endoplasmic reticulum (ER) forms membrane contact sites (MCSs) with other organelles such as mitochondria, , and in eukaryotic cells. The MCS plays a pivotal role embrane contact sites (MCSs) play an essential role in in exchanging cellular materials such as ions and . More subcellular communication by exchanging cellular materials M importantly, nonvesicular lipid trafficking occurring at the ER- and information (1, 2). Among the various endoplasmic reticulum mitochondria MCS is essential for the biogenesis of the mito- (ER)-mediated MCSs reported to date (3), the ER-mitochondria chondrial membrane. In yeast, the ER-mitochondria encounter contact site has been the most extensively studied, and an in- structure (ERMES) complex comprising the ER proteins Mmm1 volvement in ion , mitochondrial dynamics such as and cytosolic Mdm12 and the mitochondria proteins Mdm34 and membrane fission and fusion, and cooperative lipid synthesis has – Mdm10 provides a tethering force between the ER and the mi- been reported (4 9). Most importantly, lipid trafficking occurring tochondria and mediates lipid trafficking. Here, we present two at the ER-mitochondria MCS is essential for the biogenesis of the crystal structures of Mmm1 and the Mdm12–Mmm1 complex. mitochondrial membrane, since mitochondria are not connected Based on these structures, we propose the model by which the with the vesicular transport machinery, and essential lipids required Mdm12–Mmm1 complex contributes to phospholipid trafficking for the composition of mitochondrial membrane must therefore be at the ER-mitochondria MCS. supplied directly from the ER (10–12). Formation of the MCS is the result of direct interaction between Author contributions: C.L. designed research; H.J., J.P., and C.L. performed research; H.J., protein components located at two distinct subcompartments to be Y.J., and C.L. analyzed data; and H.J. and C.L. wrote the paper. adjoined. In yeast, ER-mitochondria contact sites are primarily The authors declare no conflict of interest. mediated by the ER-mitochondria encounter structure (ERMES) This article is a PNAS Direct Submission. complex that comprises four proteins: the cytosolic component Published under the PNAS license. mitochondrial distribution and morphology 12 (Mdm12); the ER Data deposition: The atomic coordinates and crystallographic structure factors have been membrane protein maintenance of mitochondrial morphology 1 deposited in the Protein Data Bank www.pdb.org (PDB ID codes 5YK6 and 5YK7). (Mmm1); and two mitochondria outer membrane proteins, 1To whom correspondence should be addressed. Email: [email protected]. Mdm34 and Mdm10 (13). Additionally, mitochondria anchoring This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2+ Gem1, a Ca -binding small GTPase, directly associates with the 1073/pnas.1715592114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1715592114 PNAS Early Edition | 1of10 Downloaded by guest on September 24, 2021 Previously, we determined the crystal structure of Saccharo- myces cerevisiae Mdm12 at 3.1 Å resolution and revealed that Mdm12 forms a dimeric SMP structure that binds phospholipids inside a hydrophobic channel, with a preference for glycer- ophospholipids harboring a positively charged head group (20). Another study determined a 17 Å resolution electron microscopy (EM) structure of the Mdm12–Mmm1 (SMP domain) complex, revealing an elongated tubular structure with an Mdm12-Mmm1- Mmm1-Mdm12 arrangement (19, 35). Despite these structure studies, the molecular-level mechanism by which the SMP do- mains of Mdm12, Mmm1, and Mdm34 are directly organized and facilitate phospholipid trafficking without consuming energy at the ER-mitochondria contact site remains unknown. Additionally, exactly how Mmm1, an ER component of the ERMES complex, recognizes specific phospholipids in the ER membrane remains elusive, as does the mechanism by which phospholipids selected by Mmm1 are transported into Mdm12, as a direct binding partner of the ERMES complex. In the present study, we determined crystal structures of the Mmm1 SMP domain and the Mdm12–Mmm1 binary complex, and discuss the resultant molecular-level insight into how the Mmm1 SMP domain contributes to the organization of the ERMES components, as well as phospholipid trafficking. Results Structure Determination of Mmm1. The Mmm1 protein is predicted to comprise a single transmembrane domain near its N terminus Fig. 1. Domain structure and direct interaction of Mmm1 and Mdm12. that anchors it to the ER membrane, an unstructured region (A) Diagrams showing the domain structure of Z. rouxii Mmm1 and S. cerevisiae consisting of around 50 residues, and an SMP domain at the C Mdm12. Mmm1 has a transmembrane (TM) domain in the middle of the terminus (Fig. 1A and Fig. S1). The N-terminal region of Mmm1 is protein chain that is required for anchoring the ER membrane, and the SMP domain is at the C terminus. Full-length scMdm12 covers the overall SMP located in the ER lumen, while the SMP domain is localized in the domain. The Mmm1 construct used in this study is indicated with an arrow and directly interacts with Mdm12, a cytosolic component (Z. rouxii Mmm1 residues 190–444, referred to as zrMmm1). To obtain of the ERMES complex. Despite significant effort to purify Mmm1 diffraction-quality crystals of the Mdm12–Mmm1 complex, two unstructured proteins, size-exclusion chromatography (SEC) experiments re- regions were omitted in the scMdm12 construct (Δ74–114 and Δ183–211, vealed that the SMP domain of S. cerevisiae Mmm1 (scMmm1) referred to as scMdm12Δ). (B) SEC profiles of scMdm12Δ (green), zrMmm1 aggregated in solution unless in a complex with Mdm12 (20). Ex- (black), and complexes of zrMmm1 and scMdm12 (blue) and zrMmm1 and tensive screening for solubility and homogeneous dispersal in so- scMdm12Δ (red). Experimental details are provided in Materials and Methods. lution for Mmm1 orthologs, together with limited proteolysis Protein standards used in the experiment are indicated above the chromato- analysis, revealed that the Mmm1 SMP domain of Zygosacchar- gram. mAu, milliabsorbance unit. omyces rouxii (zrMmm1, residues 190–444) was soluble even when not complexed with Mdm12 (Fig. 1B). The SMP domain of might be involved in the twofold interface and might be struc- zrMmm1 shares 76% sequence identity with that of scMmm1. The turally similar to that of E-SYT2 based on sequence similarity zrMmm1 proteins eluted from the gel-filtration column at a vol- (20). Consistent with our prediction, the twofold interface of the ume corresponding to the molecular weight of a dimer, suggesting zrMmm1 dimer is composed of two helices in a face-to-face ar- that the recombinantly expressed zrMmm1 SMP domain forms a rangement reminiscent of that in the E-SYT2 structure (Fig. 2B, homodimer in solution. Interestingly, the SEC experiment con- interface I and Fig. S3A). In particular, three hydrophobic resi- firmed that zrMmm1 was able to interact with scMdm12 when dues (Leu219, Trp221, and Phe222) stabilize the twofold axis coexpressed in Escherichia coli cells despite the organismal dis- through van der Waals interactions. crepancy (Fig. 1B). Diffraction-quality crystals of zrMmm1 were Upon comparing the SMP domains of E-SYT2 and Mdm12, it grown in the P3 21 space group at 4 °C over a period of 1 wk, and 2 was immediately apparent that two extra structural elements absent the structure was solved using selenomethionine-substituted crys- in the Mdm12 and E-SYT2 domains are present at the N and C tals by the single-wavelength anomalous dispersion method (Fig. B S2). The final model of zrMmm1 was refined with data from native termini of zrMmm1 (Fig. 2 and Fig. S3). These structural ele- crystals to 2.8 Å resolution. ments presumably make an important contribution to the tight association between subunits of the zrMmm1 dimer, since over 2 Structure of the zrMmm1 SMP Domain. Crystals of zrMmm1 con- 3,400 Å of solvent-accessible surface area is buried upon self- α α tained one zrMmm1 molecule in the asymmetric unit. However, association. The N terminus of zrMmm1 adopts an -helix ( 1) zrMmm1 forms a tight dimer with a crystal symmetry-related and a well-ordered loop that contacts the head region of the other molecule via a twofold rotation arrangement. The dimeric or- molecule of the dimer (interface II). In particular, the N-terminal ganization of zrMmm1 was confirmed by previous biochemical helix comprising residues 196–207 wraps around the twofold axis experiments, and is consistent with other SMP domain structures helix of the opposing molecule in an antiparallel domain-swapped (20, 36–38). Overall, the dimeric zrMmm1 SMP structure re- manner (Fig. 2B, interface II). The highly conserved C terminus of sembles a compact diamond with dimensions of 50 × 60 × 120 Å, zrMmm1 exhibits a long, extended loop that crosses over the two and each component consists of four helices and six extended molecules and essentially mediates the self-association of the and twisted antiparallel β-strands that assemble into a typical zrMmm1 dimer, as well as phospholipid binding (Fig. 2 B and C, SMP structure with an extended hydrophobic channel (Fig. 2A interface III). In more detail, the extended loop consisting of res- and Figs. S1 and S2). In a previous study, we suggested that the N idues 425–432 forms an antiparallel β-strand–like strap structure terminus (residues 198–214) of the Mmm1 SMP domain dimer that zips up the opposing twofold central helices, and eventually

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Fig. 2. Crystal structure of the zrMmm1 SMP domain. (A) Ribbon diagrams of zrMmm1 viewed in three orientations. The crystal structure of the SMP domain of zrMmm1 was determined by Se single-wavelength anomalous dispersion phasing and refined to 2.8 Å resolution. The protein adopts a dimeric SMP structure consisting of four helices and six strands in each monomer. Phospholipids bound to zrMmm1 are shown in black stick representation. Four dimeric interfaces for self-association are highlighted with black boxes. (B) Close-up view of the highlighted boxes (interfaces I–IV). Key residues that contribute to the self-association of zrMmm1 are shown in ball-and-stick representation. Oxygen and nitrogen atoms are colored red and blue, respectively. Yellow dotted lines indicate in- termolecular H-bonds. (C) Molecular surface view of zrMmm1. The surface is colored according to sequence conservation from white (variable) to dark purple (conserved) as calculated by the Consurf server (consurf.tau.ac.il) (42) using 35 different yeast orthologs. To show the orientation of zrMmm1, one molecule of the zrMmm1 dimer is drawn in ribbon representation. Highly conserved regions indicated by dotted circles are essential for self-association or interaction with Mdm12.

covers the concave surface at the center of the dimeric SMP do- electron density, we concluded that two glycerophospholipids were main (Fig. 2B, interface III). This loop also contains the absolutely bound to each zrMmm1 molecule in two distinct regions: One conserved Trp430 and Arg432 residues that are essential for the phospholipid binds at the dimeric interface (proximal), and the recognition of phospholipids, as discussed below. Additionally, the other molecule is located in the middle (distal) part of the SMP C terminus of zrMmm1 adopts a short 310 helix (residues 433–435), channel. As mentioned above, the zrMmm1 dimer formed from followed by antiparallel β-strands, and is incorporated between symmetry-related molecules in the crystal, and the two phospho- β5 and an 11-residue loop (residues 347–357) from the opposing lipids superimposed precisely over the two molecules of the molecule of the dimer through the formation of an extensive zrMmm1 dimer, suggesting that the phospholipids are specifically hydrogen-bonding network (Fig. 2B,interfaceIV). recognized by zrMmm1 and were not the result of nonspecific In summary, the extensive interfaces that are lacking in E-SYT2 binding. The head groups of two glycerophospholipids are located and Mdm12 provide the driving force for the tight self-association within a concave surface generated by helices α2–α4, and are observed in the zrMmm1 dimer. Consistently, SEC and native solvent-exposed and disordered in the structure, suggesting that PAGE revealed that the dynamic distribution between monomer zrMmm1 does not possess clear selectivity for particular phos- and dimer observed for Mdm12 and the SMP domain of E-SYT2 pholipids, consistent with Mdm12 and E-SYT2 (20, 38) (Fig. 3 B was not a feature of zrMmm1 (20). and C and Fig. S3). However, unlike in other SMP domain pro- teins, the phosphate group and carboxyl oxygen of the distal The zrMmm1 Dimer Binds Glycerophospholipids. The crystal structure phospholipid can be clearly seen in the structure, and are system- revealed that recombinant zrMmm1 expressed in con- atically coordinated by the conserved Arg253, Arg415, Trp411, tained glycerophospholipids bound in the hydrophobic channel Trp430, Arg432, and Ser433 through an extensive hydrogen- formed from the SMP domain (Fig. 3A). Based on the observed bonding network (Fig. 3C). Among these, three residues (Trp430,

Jeong et al. PNAS Early Edition | 3of10 Downloaded by guest on September 24, 2021 Fig. 3. zrMmm1 binds to glycerophospholipids. (A, Left) Overall structure of zrMmm1 (gray) bound to two phospholipids (black) viewed from the concave surface of the SMP domain. One molecule of zrMmm1 binds two phospholipids in two distinct regions, referred to as proximal and distal phospholipids (details are provided in the main text). Highly conserved C-terminal loops in the zrMmm1 dimer that are important for specific and tight lipid conjugation are colored yellow and blue. (A, Right) Molecular structures of the two phospholipids bound to zrMmm1 are shown with Fo-Fc electron density difference maps calculated in the absence of phospholipids (2.8 Å resolution, contoured at 2.0 σ). (B) Electrostatic surface representation of zrMmm1 viewed in the same orientation as in A.The electrostatic potential was calculated with the APBS program (39), and colored from −3(red)to+3(blue)kT/e(k,Boltzmann’s constant; T, temperature; e, charge of an electron). (C) Ribbon diagram showing a close-up view of the coordination of bound phospholipids (black) by the SMP domains of the zrMmm1 dimer (blue and yellow). The dimeric organization of zrMmm1 is clearly essential for the specific interactions with the phosphate ion of the distal phospholipid.(D)Invitro phospholipid displacement experiment using fluorescently labeled NBD-PE (details are provided in Materials and Methods). (Left and Center)NBD-PEpreloaded His-zrMmm1 was incubated with natural phospholipid ligands (PA, PC, PE, PG, and PS) and nonphospholipid ligands (CER, ; CH, ; EG,er- gosterol) at increasing concentrations, and the quantity of NBD-PE displaced by natural ligands was measured as the diminishment in NBD-PE fluorescence. (Right) Graph indicates quantification data. Experiments were performed in triplicate. Means ± SD are shown. (E) To probe interactions between wild-type (WT) or mutant (R379E, W411A, R415E, W430A, and R432E) zrMmm1 and phospholipids, proteins indicated in each lane were incubated with NBD-PE for 2 h on ice. Mixtures were separated by blue native PAGE, and binding was analyzed by Coomassie staining (Top) and fluorescence detection (Bottom).

Arg432, and Ser433) are from the opposing molecule in the zrMmm1 could be easily displaced by phosphatidylglycerol (PG), dimer, suggesting that lipid coordination in zrMmm1 requires (PA), PS, or phosphatidylcholine (PC), but only homodimerization. relatively weakly by PE, even at high concentrations (Fig. 3D). To examine if zrMmm1 shows preferential binding to certain However, the NBD-PE on Mmm1 could not be displaced by the phospholipids in solution, we performed lipid displacement ex- nonphospholipid cholesterol, ergosterol, or ceramide, even at high periments using 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine- concentrations (Fig. 3D). Based on these results, we conclude that N-(7-nitro-2-,3-benzoxadiazol-4-yl) (NBD)-PE, as reported in our zrMmm1 can bind efficiently to any . A pre- previous study (20). First, we confirmed the binding between vious structural study suggested that Mdm12 binds preferentially NBD-PE and purified zrMmm1 using native PAGE and fluores- to PC or PE, both of which have a positively charged head group cence detection (Fig. 3D), and found that NBD-PE bound to in common, via their negatively charged surfaces (20). Analysis of

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1715592114 Jeong et al. Downloaded by guest on September 24, 2021 the electrostatic surface potential of zrMmm1 using the Adaptive Architecture and Organization of the scMdm12Δ–zrMmm1 Complex. The PNAS PLUS Poisson–Boltzmann Solver (APBS) program (39) revealed a overall structure of the scMdm12Δ–zrMmm1 complex closely re- strong positively charged region in the vicinity of the bound sembles the EM structure described in a previous study (19) (Fig. phospholipid head group (Fig. 3B). Unlike Mdm12, the positively S4A). The scMdm12Δ–zrMmm1 complex adopts an elongated charged residues of zrMmm1 might be critically responsible for curved and tubular structure with dimensions of 60 × 65 × 210 Å. screening phospholipids themselves, not for the selection of cer- The zrMmm1 dimer is located at the center, with scMdm12Δ tain head groups of phospholipids. monomers bound at each end (Fig. 4A and Fig. S4A). Consistent Δ Next, we mutated key residues involved in lipid coordination with the previously reported model (19), scMdm12 and zrMmm1 and measured binding between zrMmm1 mutants and NBD-PE are organized in a head-to-tail manner, with the N terminus of Δ using blue native PAGE and fluorescence methods. As shown in scMdm12 (referred to as the head) that is proximal to the dimeric Fig. 3E, R415E, W411A, and W430A variants completely lost interface in the scMdm12 dimer associating with the distal end (re- the ability to bind NBD-PE, while the negative control R379E ferred to as the tail) of the homodimeric interface of the zrMmm1 SMP domain. The interaction between scMdm12Δ and zrMmm1 could still bind NBD-PE. Interestingly, two bands consistent with 2 the monomer and dimer of zrMmm1 were observed with the appearstobestrong,andburies1,012Å of surface-accessible sur- face area. The truncated residues of the unstructured loop and R415E and W430A mutants, supporting our structural analysis proline-rich region of Mdm12 are not involved in the interaction. In and conclusion that self-association of zrMmm1 is required for the crystal structure of Mdm12 alone, the N terminus (residues 1–7) lipid conjugation, and suggesting that lipid binding might en- adopts a β-strand that is involved in self-association by forming hance the stability of the dimeric form. a domain-swapped structure with the opposing molecule of the di- mer (20). However, no such conformation of Mdm12 was observed Structure Determination of the Mdm12–Mmm1 Complex. Mmm1 in the complex structure. Rather, the N terminus of scMdm12Δ specifically interacts with the Mdm12 component of the ERMES forms an extended loop structure and lies adjacent to the β2strand complex (19, 20). In our previous study, we proposed a putative Δ – of scMdm12 itself. model for the Mdm12 Mmm1 complex involving dimerization via The highly conserved β2andβ3 strands, the extended hairpin the SMP domains in a tail-to-tail manner. In this model, the loop[referredtoastheguideloop(G-loop)]generatedbetween conserved long C-terminal helices of the SMP domains lie adja- β2andβ3, and the α4 helix of zrMmm1 contribute to interactions cent to each other in a twofold rotation arrangement, resulting in with the β2andβ3 strands of scMdm12Δ (Fig. 4B). In particular,

an extended arch-shaped structure (20). However, one of the the hydrophobic amino Leu315, Leu317, Leu327, Ile388, and BIOCHEMISTRY concerns raised from this model was the lack of direct evidence for Ile397 in zrMmm1 form extensive and coordinated nonpolar con- the tail-to-tail junction, and contacts between the self-associated tacts with the side chains of Phe3, Ile5, Leu56, Ile59, Ile119, Mdm12 molecules could be an artifact of crystallization (i.e., the Phe121, and Cys170 of scMdm12Δ (Fig. 4B). In addition, Lys399 of result of crystal contacts rather than physiologically relevant mo- zrMmm1 forms a salt bridge and H-bonds with the side chain of lecular interfaces). Additionally, the potential interface between Asp61 and the main chain of Asp118 of scMdm12Δ.Toconfirm Mdm12 and Mmm1 in this model is exposed to solvent, suggest- whether these residues are involved in the interaction, we gener- ing that it is energetically unfavorable for hydrophobic glycer- ated a series of zrMmm1 mutants and scMdm12 proteins (with ophospholipids to cross the solvent region in the Mdm12 and GST fused at the N terminus of zrMmm1) and examined their Mmm1 interface. binding ability using GST pull-down experiments. Single-residue To further investigate how phospholipids could be transferred mutants of scMdm12 (L56S, I59S, I119S, and F121S) lost appre- through the SMP domains of Mdm12 and Mmm1, we determined ciable affinity for zrMmm1 (Fig. 4C). Likewise, single-site mutants the crystal structure of the Mdm12–Mmm1 complex. Initially, we of zrMmm1 (L315S or L327S) interacted with scMdm12 in a less obtained crystals of the S. cerevisiae Mdm12–Mmm1 complex and stable manner (Fig. 4D). Furthermore, to confirm the effect of the hybrid complex of scMdm12–zrMmm1, but all were of low crys- L315S mutation in solution, we titrated purified native and L315S tallographic quality. Through extensive screening, we eventually mutant tag-free zrMmm1 proteins with purified scMdm12 over a obtained diffraction-quality crystals of truncated scMdm12Δ,in wide protein concentration range and analyzed their interactions which both the unstructured loop (residues 74–114) and proline- using native PAGE. As shown in Fig. S4B, wild-type zrMmm1 rich region (residues 184–211) were excluded, in complex with interacted with scMdm12 and formed a heterotetramer in a zrMmm1 (Fig. 1A). The ability of scMdm12Δ to interact with concentration-dependent manner, while the L315S mutant did not zrMmm1 was assessed by SEC experiments (Fig. 1B). However, interact with scMdm12 at even higher concentrations, suggesting – crystals only diffracted to low resolution (∼5Å).Toovercomethis, that the observed hydrophobic contacts are critical for the Mdm12 we attempted dehydration of crystals using a higher percentage of Mmm1 interaction. precipitant, and the diffraction quality was dramatically improved The scMdm12Δ–zrMmm1 Complex Has an Extended Hydrophobic (details are provided in Materials and Methods). Dehydrated crystals Δ– Tunnel Mediating Lipid Trafficking. Structural comparison between of the scMdm12 zrMmm1 complex diffracted to 3.8 Å synchro- zrMmm1 and scMdm12 alone, and as part of the scMdm12Δ– tron radiation, and the structure was determined by the molecular zrMmm1 complex, revealed that the structure of zrMmm1 was replacement method. Crystals contained one heterotetramer orga- Δ Δ changed slightly upon complex formation. Interestingly, the nizedinanscMdm12 -zrMmm1-zrMmm1-scMdm12 arrange- structural changes appear to be functionally relevant regarding ment in the asymmetric unit (Fig. 4A). The Mdm12 modification phospholipid trafficking between the two distinct SMP domains. needed for crystallization did not affect the overall structure or First, the G-loop of zrMmm1 undergoes a conformational change binding to Mmm1 compared with wild-type Mdm12 (rmsd of 1.5 Å to form a more extended form that can plug into the scMdm12Δ for all Cα atoms). The overall conformation of zrMmm1 and head region and completely covers the solvent-exposed concave scMdm12Δ was not significantly changed upon formation of the surface of scMdm12Δ (Fig. 5A). Second, the β4strandof complex (rmsd of 0.9 Å and rmsd of 1.5 Å, respectively). No zrMmm1 is extended by two residues (Leu387 and Ile388) in the apparent electron density corresponding to the hydrocarbon complex, and these residues are part of a flexible loop and are chain of glycerophospholipids was observed in the complex solvent-exposed in the structure of zrMmm1 alone. By interacting structure except for the phosphate group of phospholipids, but with scMdm12Δ, Ile388 is projected inward toward the center of this might be due to the relatively low resolution of the complex the SMP domain and contributes to the formation of a hydro- structure or to treatments such as crystal dehydration. phobic boundary at the junction of the two SMP domains (Fig. 5B).

Jeong et al. PNAS Early Edition | 5of10 Downloaded by guest on September 24, 2021 Fig. 4. Overall architecture of the scMdm12Δ–zrMmm1 complex. (A) Figures showing the overall architecture of the scMdm12Δ (green)–zrMmm1 (blue) complex. The structure of the scMdm12Δ–zrMmm1 complex was determined by the molecular replacement method and refined to 3.8 Å resolution. The 2Fo-Fc electron density map (Left, calculated with data to 3.8 Å resolution and contoured at 1.0 σ) and the surface representation of the crystallographic asymmetric unit of the scMdm12Δ–zrMmm1 complex (Right) are shown. Phosphate ions are shown as ball-and-stick models with red for oxygen and orange for atoms. (B) Binding interface between zrMmm1 and scMdm12Δ in three orientations. Residues involved in the interaction are shown in ball-and-stick representation. (C)Role of scMdm12 residues in the interaction with zrMmm1 assessed through GST pull-down experiments using scMdm12 mutants (L56S, I59S, I119S, and F121S). (D)SDS/ PAGE showing the results of a reciprocal test of the effect of mutations in zrMmm1 (L315S and L327S) on the interaction with scMdm12. WT, wild type.

Third, the conserved loop formed between β4andα4, which are The scMdm12–zrMmm1 Complex Binds All Glycerophospholipids Except well ordered in the structure of zrMmm1 alone, becomes disor- for PE in Vitro. To identify differences in binding priority to phos- dered upon forming a complex with scMdm12Δ.Inparticular, pholipids between the scMdm12–zrMmm1 complex and zrMmm1 three hydrophilic residues (Arg391, Ser392, and Lys393) are not or scMdm12 alone, we performed a lipid displacement experiment visible in the scMdm12Δ–zrMmm1 complex (Fig. 5B). Finally, the using the scMdm12–zrMmm1 complex. Interestingly, NBD-PE α4 helix of zrMmm1 and the loop formed between α3andβ1are bound to the scMdm12–zrMmm1 complex could be displaced pushed outward, generating a wider space inside the cavity that only by PA, PG, PC, or PS (Fig. 6A). In the case of PA, high might be important for phospholipid trafficking (Fig. 5 C and concentrations resulted in band shifts above those of the NBD-PE D). Taken together, the formation of the scMdm12Δ–zrMmm1 preloaded scMdm12–zrMmm1 complex alone on native PAGE. complex generates a continuous hydrophobic tunnel ∼210 Å long No such changes have been observed using NBD-PE–preloaded through the elongated SMP domains of scMdm12Δ and zrMmm1, scMdm12 alone (20). However, high concentrations of PA also which could conceivably translocate phospholipids harboring resulted in similar band shifts of NBD-PE–preloaded zrMmm1 nonpolar hydrocarbon chains between two components without alone, indicating that PA binding to zrMmm1 might affect the consuming energy (Fig. 5E). These results strongly indicate that overall conformation of zrMmm1 or the scMdm12–zrMmm1 the Mdm12–Mmm1 complex acts as a lipid-transferring vehicle in complex. addition to tethering molecules to physically connect two distinct One of the most striking differences between zrMmm1 and the subcompartments. scMdm12–zrMmm1 complex was the absence of scMdm12-zrMmm1

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Fig. 5. Direct association of zrMmm1 and scMdm12Δ generates a hydrophobic tunnel for phospholipid trafficking. (A) Ribbon diagram showing superposition of zrMmm1 (yellow) and the scMdm12Δ (green)–zrMmm1 (blue) complex. To analyze structural changes in zrMmm1 upon association with scMdm12Δ, the structure of zrMmm1 was aligned with the zrMmm1 structure in the scMdm12Δ–zrMmm1 complex. The scMdm12Δ is shown in surface representation. The G-loop of zrMmm1 undergoes conformational changes following interaction with scMdm12Δ, forming an extended structure that covers the solvent-exposed region of scMdm12Δ. Residues of zrMmm1 undergoing this structural reorganization are shown, and their directions are indicated with red arrows. (B)Structuralchangesin zrMmm1 occurring upon association with scMdm12Δ further highlighted (more information is provided in the main text) in a diagram colored the same as in A. The dotted line indicates zrMmm1 residues that become disordered upon forming the complex. (C, Right) Direct association of zrMmm1 and scMdm12Δ moves the α4helixofzrMmm1by∼10° outward, vacating enough space to accommodate and transfer phospholipids. Phospholipids bound to zrMmm1 are shown in surface-filling representation. The red arrow indicates the putative pathway of phospholipids from zrMmm1 to scMdm12Δ.(C, Left)Ribbondiagramscompare the overall structure of zrMmm1 in the apo (yellow) and complexed (blue) forms viewed from the left side of the figure (C, Right). Loops, including Tyr261, in the complexed form are shifted outward, generating an open space in the process. The scMdm12Δ is omitted for clarity. The overall color scheme is the same as in A.(D) Structures of zrMmm1 in the apo (yellow) and complexed (blue) forms viewed from the right side of the picture (C, Right). (E) Overall structure of the scMdm12Δ–zrMmm1 complex shown in meshed line (Top)andribbon(Bottom)representations.(Top) Red mesh representing hydrophobic amino acids inside the tunnel was superimposed on the figure. (Bottom) Channel (cavity) through the scMdm12Δ–zrMmm1 complex was analyzed by Mole 2.0 (43), and is shown in black tubule representation. Black arrows indicate the putative pathway for phospholipid trafficking.

binding to PE (Fig. 6A). Even though both scMdm12 alone and outside mitochondria via the Kennedy pathway might not be zrMmm1 alone bound to PE with noticeable efficiency (20) efficiently transferred to mitochondria for unknown reasons (Figs. 3D and 6B), the scMdm12–zrMmm1 complex did not bind (40). Consistent with this, the scMdm12–zrMmm1 complex did PE at all, suggesting that the association between scMdm12 and not engage in PE binding in vitro. zrMmm1 affects the binding preferences of zrMmm1 and PS transfer to mitochondria is required for the synthesis of PE scMdm12 to phospholipids. Although the tests were performed in mitochondria. Because scMdm12 alone could not bind PS (20) using purified proteins in vitro, these results could have impor- (Fig. 6B), we inferred that the PS that displaced NBD-PE from tant biological implications. The PE component of the mito- scMdm12 in the scMdm12–zrMmm1 complex might have been chondrial membrane might not be directly transferred from the directly transferred from zrMmm1. To verify this, we generated ER but might be synthesized within the mitochondrial matrix via an Y261W mutant of zrMmm1. The Y261 residue is located at the conversion of PS to PE. Furthermore, the PE generated the interface between zrMmm1 and scMdm12 and is involved in

Jeong et al. PNAS Early Edition | 7of10 Downloaded by guest on September 24, 2021 Fig. 6. The scMdm12–zrMmm1 complex does not bind PE in vitro, and acts as a lipid transfer module. (A) In vitro phospholipid displacement experiments using the scMdm12–zrMmm1 complex. NBD-PE–preloaded scMdm12–zrMmm1 complexes were mixed with increasing concentrations of phospholipids (PA, PC, PE, PG, and PS). Decreasing fluorescence was used to measure NBD-PE displacement by each phospholipid. (Left) Fluorescence and Coomassie staining of clear-native PAGE gels are shown side by side. (Right) Graph shows quantification data. Experiments were performed in triplicate, independently. Means ± SD are given. (B, Left) Schematic diagram shows possible routes for phospholipid access to Mdm12 or Mmm1 in the Mdm12–Mmm1 complex. The table below shows a summary of the results of the phospholipid displacement experiment using Mmm1, Mdm12–Mmm1 complex (from this study), and Mdm12 (20). The symbols X, △, and ○ indicate that the fluorescence reduction rate is within the range of 0–35%, 35–70%, and 70–100% at 250 μM, respectively, of each phospholipid. (B, Right) Ribbon diagram highlights the role of the zrMmm1 Y261 residue at the interface between scMdm12Δ and zrMmm1. (C) SEC analysis shows that the Y261W mutant of zrMmm1 can still associate with scMdm12. Molecular weight standards are indicated above the chromatogram. mAu, milliabsorbance unit. (D) In vitro phospholipid displacement experiment with the scMdm12–zrMmm1 complex (wild-type and Y261W mutant). The graph indicates the concentration of a phospholipid required to reduce the NBD-PE fluorescence signal by 50%. The bar graph shows means ± SD (n = 3). (E) Schematic representation highlights the role of the SMP domain in phospholipid transport. The SMP domains in the two distinct subunits directly associate with each other, generating a successive hydrophobic tunnel through which phospholipid transfer can occur from one subunit to the other.

generating a hydrophobic channel. However, the residue does SMP domains in the scMdm12–zrMmm1 complex generates a not directly contribute to the interaction between scMdm12 and hydrophobic tunnel for lipid trafficking. zrMmm1 (Figs. 5C and 6B). We hypothesized that the conver- sion of Tyr to Trp would sterically hinder the transfer of phos- Discussion pholipids between zrMmm1 and scMdm12. As expected, the SMP domains in ERMES and tubular lipid-binding superfamily mutation did not affect the association between scMdm12 and complexes are believed to have a common role in binding and zrMmm1(Fig. 6C), and PS binding by the zrMmm1 (Y261W) transferring lipids (41). However, molecular recognition of specific mutant was similar to that of wild-type zrMmm1 (Fig. S5). phospholipids by SMP domains is not conserved among SMP- However, in contrast to the wild type, the NBD-PE bound to the containing proteins. For example, scMdm12 has a binding pref- zrMmm1(Y261W)–scMdm12 complex was slowly displaced by erence for phospholipids harboring positively charged head PS (Fig. 6D), suggesting that the bulky side chain of Trp sterically groups, while the SMP domain of zrMmm1 broadly binds to most impeded PS transfer from zrMmm1 to scMdm12 (Fig. 6E). We phospholipids, although zrMmm1 preferentially binds to PS, PA, also tested whether the mutation affected the displacement of PG, and PC. In addition, our previous work revealed that NBD-PE from the zrMmm1(Y261W)–scMdm12 complex by PC scMdm12 binds one molecule of phospholipid (20), while the and PG, and observed that PC, but not PG, resulted in slightly zrMmm1 SMP domain binds two phospholipids in distinct regions slow displacement (Fig. 6D). Since scMdm12 alone could effi- (Fig. S3B). Interestingly, the phosphate group of the distal phos- ciently bind to PC and PG unlike PS (20) (Fig. 6B), the effect of pholipid is specifically coordinated by conserved residues in the mutation might not be significant in vitro. In summary, from zrMmm1 (Fig. 3C). Specifically, two pairs of Arg-Trp residues these observations, we confirmed that the direct association of (Arg415/Trp411 and Arg432/Trp430 from the opposing molecule

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1715592114 Jeong et al. Downloaded by guest on September 24, 2021 of the zrMmm1 dimer), which are absolutely conserved among Mdm12–Mmm1 interaction (Fig. 4A). At present, it remains dif- PNAS PLUS other Mmm1 orthologs, form an extensive H-bonding network ficult to test these models because the interaction is likely to be with the phosphate ion and carboxyl oxygen of the phospholipid transient. Interestingly, the scMdm12Δ–zrMmm1 structure dem- (Fig. 3C). From this observation, we proposed that the Arg and onstrates that it is possible to generate a continuous hydrophobic Trp residues act as a filter for screening phospholipids among the tunnel through both the head and tail of Mdm12 (Fig. 5E), sug- pool of cellular lipids. This represents a unique feature of gesting that the head and tail of Mdm12 might interact directly Mmm1 because most SMP domains bind hydrocarbon chains of with the head of Mdm34. Future work is required to address ex- phospholipids through nonpolar contacts with hydrophobic resi- actly how the SMP domain of Mdm34 is organized in the Mmm1– dues inside the cavity of the SMP domain. Mdm12–Mdm34 ternary complex. Regarding phospholipid trafficking at the ER-mitochondria con- In conclusion, the Mdm12–Mmm1 complex establishes a tact site, it is well established that PC is synthesized from PS via PE molecular basis for protein-mediated MCSs between the ER and through the action of two enzymes that are distinctly located in the mitochondria, and for phospholipid trafficking through the ER and mitochondria. The conversion of PS to PE is catalyzed by ERMES complex. enzymes resident in mitochondria, whereas PA, an important in- termediate in the formation of PG and in mitochondria, Materials and Methods is synthesized in the ER (11). PS, PA, and PG must therefore be Plasmid Construction. The DNA fragment encoding the SMP domain of Mmm1 transferred from the ER, their site of synthesis, to mitochondria. (Z. rouxii, residues 190–444) was generated by PCR amplification from ge- Furthermore, PC synthesized in the ER must be eventually trans- nomic DNA and cloned into the pET28b-SMT3 expression vector with BamHI Δ – located to mitochondria for maintenance of membrane integrity. and SalI restriction enzymes. To construct scMdm12 , residues 74 114 and residues 183–211 from full-length Mdm12 were substituted to GGSGG (E73- Because Mmm1 is the only ER resident protein among ERMES GGSGG-S115) and GG (D182-GG-S212), respectively, and cloned into the components, and since Mmm1 might be involved in phospholipid pCDF-duet vector with NdeI and XhoI. All mutants were generated by PCR- selection from the ER, the specific and favored recognition of based mutagenesis, and mutations were confirmed by DNA sequencing. phospholipids by Mmm1 might help to facilitate efficient lipid traf- ficking. In this study, we structurally and biochemically demonstrated Protein Expression and Purification. All proteins in this study were expressed by that zrMmm1 alone and the scMdm12–zrMmm1 complex prefer- transforming the expression plasmids into E. coli BL21 (DE3) bacterial cells. Cells ∼ entially bind to phospholipids. This apparent selective extraction of were grown to an OD600 nm of 0.7 at 37 °C with vigorous shaking and in- phospholipids, facilitated by the surface charge and phospholipid duced overnight at 18 °C with 0.3 mM isopropyl-β-D-thiogalactoside. Cells were × filter of Mmm1, might be critical to the initiation of cooperative collected by centrifugation at 3,200 g for 15 min; resuspended in buffer A BIOCHEMISTRY phospholipid synthesis at ER-mitochondria contact sites. containing 25 mM sodium phosphate (pH 7.8), 400 mM sodium chloride, and 10 mM imidazole; and flash-frozen in liquid nitrogen for later use. The The proximal surfaces of membrane proteins are often posi- zrMmm1 proteins were purified by Ni2+-immobilized metal affinity chroma- tively charged, and we therefore suggest that the positively 2+ tography (Ni -IMAC). His6-SMT3 tags were removed by adding Ulp1 protease charged concave inner surface in the SMP domain of zrMmm1 at a ratio of 1:1,000 (wt/wt), and proteins were dialyzed overnight against might interact closely with the ER membrane. The concave bufferBcomprising25mMTris·HCl (pH 7.5), 150 mM sodium chloride, and structure of zrMmm1 might complement membrane curvature in 5mMβ-mercaptoethanol at 4 °C. Digested proteins were passed through an + terms of shape and size. In addition, the adjacent circumference of Ni2 -chelating column a second time to remove SMT3 tags and undigested a positively charged patch composed of hydrophobic residues, protein, followed by SEC with a Superdex 200 (16/60) column (GE Healthcare) including Y245, W238, P354, P357, and Y406, with the side chains preequilibrated with buffer C comprising 25 mM Tris·HCl (pH 7.5), 150 mM of these residues exposed to the surface of zrMmm1, indicates that sodium chloride, and 5 mM DTT. For the scMdm12Δ–zrMmm1 complex, pET28b-SMT3-zrMmm1 and pCDF- these residues might play a role in tight docking to the ER duet-scMdm12Δ plasmids were simultaneously transformed into E. coli BL21 membrane (Fig. S6 A and B). Interestingly, we observed that (DE3) cells. The scMdm12Δ–zrMmm1 complex proteins were purified using unlike the head groups of phospholipids bound to Mdm12, which Ni2+-IMAC. After Ulp1 digestion, proteins were further purified by HiTrap Q are distal from the concave surface of Mdm12, the head groups of HP (GE Healthcare) and Superdex 200 columns in buffer C. Purified zrMmm1 phospholipids bound to zrMmm1 project into the concave surface and scMdm12Δ–zrMmm1 complex proteins were concentrated to 12.5 mg/mL of zrMmm1 (Fig. S6C). Moreover, the concave surface in the and 5 mg/mL, respectively, using Amicon ultra-15 centrifugal filters (Merck scMdm12–zrMmm1 complex precisely conforms to that generated Millipore), and were flash-frozen at −80 °C for later use. by zrMmm1, strongly supporting the possibility that the concave For selenomethionine-substituted proteins, the zrMmm1 plasmid was inner surface of zrMmm1 binds to a convex membrane region. transformed and expressed in the E. coli B834 (DE3) auxotrophic strain. Cells were grown in M9 minimal media supplemented with L-sele- Mmm1 interacts with Mdm34 through Mdm12 via relatively nomethionine, and proteins were purified as described above. Additional weak or transient interactions (19, 20). Additionally, we previously methods are described in SI Materials and Methods. suggested that the N terminus of Mdm34 might be involved in the interaction with Mdm12 (20). Based on these findings, we propose ACKNOWLEDGMENTS. We thank staff of the 5C beamline at the Pohang Ac- two putative models for the organization of the ERMES complex. celerator Laboratory for assistance with synchrotron facilities. This research was First, the N terminus of Mdm34 might interact with the N ter- supported by the Logistics Research Center (Grant 2016R1A5A1007318), the β Basic Research Program (Grant NRF-2015R1D1A1A01058016), and the Global minus of Mdm12 via -strand swapping, as shown in the Mdm12 Ph.D. Fellowship Program Grant NRF-2014H1A2A1020322 (to H.J.) from the dimer (20). Second, the head of the Mdm34 SMP domain might National Research Foundation of Korea. This work was also supported by the interact with the tail of the Mdm12 SMP domain, as shown in the Institute for Basic Science (Grant IBS-R022-D1).

1. Elbaz Y, Schuldiner M (2011) Staying in touch: The molecular era of contact 7. Lang A, John Peter AT, Kornmann B (2015) ER-mitochondria contact sites in yeast: sites. Trends Biochem Sci 36:616–623. Beyond the myths of ERMES. Curr Opin Cell Biol 35:7–12. 2. Helle SC, et al. (2013) Organization and function of membrane contact sites. Biochim 8. Csordás G, et al. (2006) Structural and functional features and significance of the Biophys Acta 1833:2526–2541. physical linkage between ER and mitochondria. J Cell Biol 174:915–921. 3. Phillips MJ, Voeltz GK (2016) Structure and function of ER membrane contact sites 9. Friedman JR, et al. (2011) ER tubules mark sites of mitochondrial division. Science 334: with other organelles. Nat Rev Mol Cell Biol 17:69–82. 358–362. 4. Kornmann B, Walter P (2010) ERMES-mediated ER-mitochondria contacts: Molecular 10. Tatsuta T, Scharwey M, Langer T (2014) Mitochondrial lipid trafficking. Trends Cell hubs for the regulation of mitochondrial . J Cell Sci 123:1389–1393. Biol 24:44–52. 5. Rowland AA, Voeltz GK (2012) Endoplasmic reticulum-mitochondria contacts: Func- 11. Flis VV, Daum G (2013) Lipid transport between the endoplasmic reticulum and mi- tion of the junction. Nat Rev Mol Cell Biol 13:607–625. tochondria. Cold Spring Harb Perspect Biol 5:a013235. 6. Kornmann B (2013) The molecular hug between the ER and the mitochondria. Curr 12. Lev S (2010) Non-vesicular lipid transport by lipid-transfer proteins and beyond. Nat Opin Cell Biol 25:443–448. Rev Mol Cell Biol 11:739–750.

Jeong et al. PNAS Early Edition | 9of10 Downloaded by guest on September 24, 2021 13. Kornmann B, et al. (2009) An ER-mitochondria tethering complex revealed by a 32. Mao K, Klionsky DJ (2013) Participation of mitochondrial fission during mitophagy. synthetic biology screen. Science 325:477–481. Cell Cycle 12:3131–3132. 14. Kornmann B, Osman C, Walter P (2011) The conserved GTPase Gem1 regulates 33. Mao K, Wang K, Liu X, Klionsky DJ (2013) The scaffold protein Atg11 recruits fission endoplasmic reticulum-mitochondria connections. Proc Natl Acad Sci USA 108: machinery to drive selective mitochondria degradation by . Dev Cell 26: 14151–14156. 9–18. 15. Murley A, et al. (2013) ER-associated mitochondrial division links the distribution of 34. Böckler S, Westermann B (2014) Mitochondrial ER contacts are crucial for mitophagy mitochondria and mitochondrial DNA in yeast. Elife 2:e00422. in yeast. Dev Cell 28:450–458. 16. Stroud DA, et al. (2011) Composition and topology of the endoplasmic reticulum- 35. AhYoung AP, Lu B, Cascio D, Egea PF (2017) Crystal structure of Mdm12 and combi- mitochondria encounter structure. J Mol Biol 413:743–750. 17. Belgareh-Touzé N, Cavellini L, Cohen MM (2017) Ubiquitination of ERMES compo- natorial reconstitution of Mdm12/Mmm1 ERMES complexes for structural studies. – nents by the E3 ligase Rsp5 is involved in mitophagy. Autophagy 13:114–132. Biochem Biophys Res Commun 488:129 135. 18. Kopec KO, Alva V, Lupas AN (2010) Homology of SMP domains to the TULIP super- 36. Beamer LJ, Carroll SF, Eisenberg D (1997) Crystal structure of human BPI and two family of lipid-binding proteins provides a structural basis for lipid exchange between bound phospholipids at 2.4 angstrom resolution. Science 276:1861–1864. ER and mitochondria. Bioinformatics 26:1927–1931. 37. Qiu X, et al. (2007) Crystal structure of cholesteryl ester transfer protein reveals a long 19. AhYoung AP, et al. (2015) Conserved SMP domains of the ERMES complex bind tunnel and four bound lipid molecules. Nat Struct Mol Biol 14:106–113. phospholipids and mediate tether assembly. Proc Natl Acad Sci USA 112:E3179–E3188. 38. Schauder CM, et al. (2014) Structure of a lipid-bound extended synaptotagmin indi- 20. Jeong H, Park J, Lee C (2016) Crystal structure of Mdm12 reveals the architecture and cates a role in lipid transfer. Nature 510:552–555. dynamic organization of the ERMES complex. EMBO Rep 17:1857–1871. 39. Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Electrostatics of nano- 21. Toulmay A, Prinz WA (2012) A conserved membrane-binding domain targets proteins systems: Application to microtubules and the ribosome. Proc Natl Acad Sci USA 98: to organelle contact sites. J Cell Sci 125:49–58. 10037–10041. 22. Kojima R, Endo T, Tamura Y (2016) A phospholipid transfer function of ER- 40. Bürgermeister M, Birner-Grünberger R, Nebauer R, Daum G (2004) Contribution of mitochondria encounter structure revealed in vitro. Sci Rep 6:30777. different pathways to the supply of phosphatidylethanolamine and phosphatidyl- 23. Nguyen TT, et al. (2012) Gem1 and ERMES do not directly affect phosphatidylserine to mitochondrial membranes of the yeast . Biochim transport from ER to mitochondria or mitochondrial inheritance. Traffic 13:880–890. – 24. Elbaz-Alon Y, et al. (2014) A dynamic interface between vacuoles and mitochondria in Biophys Acta 1686:161 168. yeast. Dev Cell 30:95–102. 41. Alva V, Lupas AN (2016) The TULIP superfamily of eukaryotic lipid-binding proteins as 25. Hönscher C, et al. (2014) Cellular regulates contact sites between vacuoles a mediator of lipid sensing and transport. Biochim Biophys Acta 1861:913–923. and mitochondria. Dev Cell 30:86–94. 42. Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N (2010) ConSurf 2010: Calculating 26. Lahiri S, et al. (2014) A conserved endoplasmic reticulum membrane protein complex evolutionary conservation in sequence and structure of proteins and nucleic acids. (EMC) facilitates phospholipid transfer from the ER to mitochondria. PLoS Biol 12: Nucleic Acids Res 38:W529–W533. e1001969. 43. Sehnal D, et al. (2013) MOLE 2.0: Advanced approach for analysis of bio- 27. Ellenrieder L, et al. (2016) Separating mitochondrial protein assembly and endo- macromolecular channels. J Cheminform 5:39. plasmic reticulum tethering by selective coupling of Mdm10. Nat Commun 7:13021. 44. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in 28. Meisinger C, et al. (2007) The morphology proteins Mdm12/Mmm1 function in the oscillation mode. Methods in Enzymology, ed Carter CW, Jr (Academic, New York), major β-barrel assembly pathway of mitochondria. EMBO J 26:2229–2239. Vol 276, pp 307–326. 29. Hobbs AE, Srinivasan M, McCaffery JM, Jensen RE (2001) Mmm1p, a mitochondrial 45. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro- outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and molecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221. required for mtDNA stability. J Cell Biol 152:401–410. 30. Meeusen S, Nunnari J (2003) Evidence for a two membrane-spanning autonomous 46. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. – mitochondrial DNA replisome. J Cell Biol 163:503–510. Acta Crystallogr D Biol Crystallogr 66:486 501. – 31. Berger KH, Sogo LF, Yaffe MP (1997) Mdm12p, a component required for mito- 47. McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Cryst 40:658 674. chondrial inheritance that is conserved between budding and fission yeast. J Cell Biol 48. Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of mem- 136:545–553. brane protein complexes in enzymatically active form. Anal Biochem 199:223–231.

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